Type VI secretion systems (T6SS) are organelles that are encoded by gene clusters present in many Gram-negative bacterial species in including Pseudomonas aeruginosa, an opportunistic pathogen of Cystic Fibrosis (CF) patients. In many bacterial organisms, T6SS are known virulence factors but recently it has been further recognized that these systems can also kill bacterial as well as eukaryotic cells. Our laboratory has shown that functionally these nanomachines transport proteins into target host cells by a novel mechanism that is analogous to bacteriophage tail contraction. This process can be visualized in real time using appropriate fluorescent reporter protein fusions. The T6SS dynamic organelle assembles and attacks prey cells by initially penetrating them with a large protein ensemble called the VgrG/PAAR/Hcp spike/ tube complex which we hypothesize is decorated with toxic effector proteins by several mechanisms. Furthermore, we have shown that P. aeruginosa can use its T6SS to counterattack and kill other T6SS+ aggressive prey organisms (such as Vibrio cholerae and Acinetobacter baylyi) that attack its cell envelope. A P. aeruginosa sensory system called TagQRST-PpkA-Fha1 regulatory cascade drives T6SS organelle assembly at the site of prey attack and also responds to the outer membrane active, cationic antibiotic polymyxin B (PxB). Therefore, we propose to investigate the following questions in the context of P. aeruginosa: 1) Will PxB activate T6SS assembly in PxB resistant P. aeruginosa mutants and will these mutants still counterattack other aggressive T6SS+ prey cells? 2) Will other membrane stress signals activate organelle assembly including exposure to host defense molecules that attack the bacterial cell envelope (such as human defensins and other toxic cationic and antimicrobial peptides)? 3) Can we use PxB activation to facilitate the large-scale purification of T6SS organelles from P. aeruginosa for structural analysis? 4) Are different PAAR and VgrG proteins required for secretion of different VgrG proteins and T6SS effectors? 5) Will a new saturation mutagenesis technique developed in our lab called Mut-seq allow us to perform a comprehensive analysis of the protein sequences required for T6SS function and allow us further to isolate dominant-negative versions of some of its essential components? 6) Can we define the genes that encode the T6SS armor which allows P. aeruginosa to resist killing by other antibacterial T6SS+ species and their effectors? In pursuing the answers to these questions we will also determine if a broad panel of P. aeruginosa strains express T6SS organelles and characterize these using state-of-the-art imaging methodologies including fluorescence microscopy and electron cryo tomography. Our studies offer novel opportunities for development of therapeutics that attack the cell envelope of P. aeruginosa, a notoriously antibiotic-resistant organism. Thus, these studies may ultimately benefit CF and other patients suffering from P. aeruginosa infections and potentially also contribute to the development of new strategies to inhibit Gram-negative bacteria including those that use the T6SS as a virulence factor.